Pressure-flow relationships for packed beds of compressible chromatography media at laboratory and production scale

Pressure drop across chromatography beds employing soft or semirigid media can be a significant problem in the operation of large-scale preparative chromatography columns. The shape or aspect ratio (length/diameter) of a packed bed has a significant effect on column pressure drop due to wall effects, which can result in unexpectedly high pressures in manufacturing. Two types of agarose-based media were packed in chromatography columns at various column aspect ratios, during which pressure drop, bed height, and flow rate were carefully monitored. Compression of the packed beds with increasing flow velocities was observed. An empirical model was developed to correlate pressure drop with the aspect ratio of the packed beds and the superficial velocity. Modeling employed the Blake-Kozeny equation in which empirical relationships were used to predict bed porosity as a function of aspect ratio and flow velocity. Model predictions were in good agreement with observed pressure drops of industrial scale chromatography columns. A protocol was developed to predict compression in industrial chromatography applications by a few laboratory experiments. The protocol is shown to be useful in the development of chromatographic methods and sizing of preparative columns.     

Automated methods for accurate determination of the critical velocity of packed bed chromatography

Knowing the critical velocity (ucrit) of a chromatography column is an important part of process development as it allows the optimization of chromatographic flow conditions. The conventional flow step method for determining ucrit is prone to error as it depends heavily on human judgment. In this study, two automated methods for determining ucrit have been developed: the automatic flow step (AFS) method and the automatic pressure step (APS) method. In the AFS method, the column pressure drop is monitored upon application of automated incremental increases in flow velocity, whereas in the APS method the flow velocity is monitored upon application of automated incremental increases in pressure drop. The APS method emerged as the one with the higher levels of accuracy, efficiency and ease of application having the greater potential to assist defining the best operational parameters of a chromatography column.     

Simulation of the dynamic packing behavior of preparative chromatography columns via discrete particle modeling

Preparative packed‐bed chromatography using polymer‐based, compressible, porous resins is a powerful method for purification of macromolecular bioproducts. During operation, a complex, hysteretic, thus, history‐dependent packed bed behavior is often observed but theoretical understanding of the causes is limited. Therefore, a rigorous modeling approach of the chromatography column on the particle scale has been made which takes into account interparticle micromechanics and fluid–particle interactions for the first time. A three‐dimensional deterministic model was created by applying Computational Fluid Dynamics (CFD) coupled with the Discrete Element Method (DEM). The column packing behavior during either flow or mechanical compression was investigated in‐silico and in laboratory experiments. A pronounced axial compression–relaxation profile was identified that differed for both compression strategies. Void spaces were clearly visible in the packed bed after compression. It was assumed that the observed bed inhomogeneity was because of a force‐chain network at the particle scale. The simulation satisfactorily reproduced the measured behavior regarding packing compression as well as pressure‐flow dependency. Furthermore, the particle Young's modulus and particle–wall friction as well as interparticle friction were identified as crucial parameters affecting packing dynamics. It was concluded that compaction of the chromatographic bed is rather because of particle rearrangement than particle deformation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:363–371, 2016     

Pore network modelling of affinity chromatography: determination of the dynamic profiles of the pore diffusivity of beta-galactosidase and its effect on column performance as the loading of beta-galactosidase onto anti-beta-galactosidase varies with time.

A three-dimensional pore network model for diffusion in porous adsorbent particles was employed in a dynamic adsorption model that simulates the adsorption of a solute in porous particles packed in a chromatographic column. The solution of the combined model yielded the dynamic profiles of the pore diffusion coefficient of beta-galactosidase along the radius of porous adsorbent particles and along the length of the column as the loading of beta-galactosidase onto anti-beta-galactosidase immobilized on the surface of the pores of the particles occurred, and, the dynamic adsorptive capacity of the chromatographic column as a function of the design and operational parameters of the chromatographic system. It was found that for a given column length the dynamic profiles of the pore diffusion coefficient were influenced by (a) the superficial fluid velocity in the column, (b) the diameter of the adsorbent particles, and (c) the pore connectivity of the porous structure of the adsorbent particles. The effect of the magnitude of the pore connectivity on the dynamic profiles of the pore diffusion coefficient of beta-galactosidase increased as the diameter of the adsorbent particles and the superficial fluid velocity in the column increased. The dynamic adsorptive capacity of the column increased as (i) the particle diameter and the superficial fluid velocity in the column decreased, and (ii) the column length and the pore connectivity increased. In preparative affinity chromatography, it is desirable to obtain high throughputs within acceptable pressure gradients, and this may require the employment of larger diameter adsorbent particles. In such a case, longer column lengths satisfying acceptable pressure gradients with adsorbent particles having higher pore connectivity values could provide high dynamic adsorptive capacities. An alternative chromatographic system could be comprised of a long column packed with large particles which have fractal pores (fractal particles) that have high pore connectivities and which allow high intraparticle diffusional and convective flow mass transfer rates providing high throughputs and high dynamic adsorptive capacities. If large scale monoliths could be made to be reproducible and operationally stable, they could also offer an alternative mode of operation that could provide high throughputs and high dynamic adsorptive capacities.     

Toward a robust model of packing and scale-up for chromatographic beds. 1. Mechanical compression

The packing of compressible biochromatographic resins at large scale suffers from a poor understanding of how column packing method, resin properties, and column geometry impact column performance. To improve understanding, we develop and evaluate a one-dimensional, continuum mechanics model of column packing by mechanical compression. We show that the model can quantitatively predict the change in bed height, applied stress, and internal axial porosity profile without adjustable parameters when the modulus and wall friction coefficients are determined independently. The model possesses theoretical relationships for wall support and resin rigidity that should enable it to describe the mechanical compression of any biochromatographic resin for any column diameter. Moreover, this framework could provide a path to analogous models for flow packing and dynamic axial compression.     

Modeling of scale-down effects on the hydrodynamics of expanded bed adsorption columns

Expanded-bed adsorption (EBA) is a technique for primary recovery of proteins starting from unclarified broths. This process combines centrifugation, concentration, filtration, and initial capturing of the proteins in a single step. An expanded bed (EB) is comparable to a packed bed in terms of separation performance but its hydrodynamics are that of a fluidized bed. Downstream process development involving EBA is normally carried out in small columns to minimize time and costs. Our purpose here is to characterize the hydrodynamics of expanded beds of different diameters, to develop scaling parameters that can be reliably used to predict separation efficiency of larger EBA columns. A hydrodynamic model has been developed which takes into account the radial liquid velocity profile in the column. The scale-down effect can be characterized in terms of apparent axial dispersion, D(axl,app), and plate number, N(EB), adapted for expanded bed. The model is in good agreement with experimental results obtained from 1- and 5-cm column diameters with buffer solutions of different viscosities. The model and the experiments show an increase of apparent axial dispersion with an increase in column diameter. Furthermore, the apparent axial dispersion is affected by an increase in liquid velocity and viscosity. Supported by visual observations and predictions from the model, it was concluded that operating conditions (liquid viscosity and superficial velocity) resulting in a bed-void fraction between 0.7 and 0.75 would provide the optimal separation efficiency in terms of N(EB).     

Analytical techniques for cell fractions. XIX. The cyclum: an automatic system for cyclic chromatography.

An automatic system, termed a Cyclum, is described which allows column chromatographic separations to be repeated precisely a large number of times. Provision is made for the adjustment during operation of parameters such as equilibration, wash, elution, and sample flow times and duration of fraction collection. The system is applicable to both analytical and preparative use in various types of column chromatography (e.g., affinity, gel filtration, ion-exchange), but has been especially developed for separations based on immunosorption.     

The effects of processing scale on the pressure drop of compressible gel supports in liquid chromatographic columns

Experimental data are given for the solid pressure distributions in liquid chromatographic columns for two commercially available agarose-based compressible gel supports. The data show that for a given liquid velocity the pressure drop increases exponentially with height from the top surface. However, beyond a threshold liquid velocity, flow instability develops rapidly and the pressure drop rises sharply as the gel matrix compresses under the applied vertical pressures. The threshold liquid velocity at the point of criticality is examined in the light of the experimental data and the numerical simulations based on a one dimensional elemental slice force balance of the gel mass. The result of this analysis shows that matrix rigidity, column diameter and bed height significantly affect the point of criticality.     

Online integrity monitoring in the Protein A step of mAb Production Processes—increasing reliability and process robustness

The purification of recombinant proteins and antibodies using large packed‐bed columns is a key component in most biotechnology purification processes. Because of its efficiency and established practice in the industry, column chromatography is a state of the art technology with a proven capability for removal of impurities, viral clearance, and process efficiency. In general, the validation and monitoring of chromatographic operations—especially of critical process parameters—is required to ensure robust product quality and compliance with health authority expectations. One key aspect of chromatography that needs to be monitored is the integrity of the packed bed, since this is often critical to achieving sufficient separation of protein species. Identification of potential column integrity issues before they occur is important for both product quality and economic efficiency. In this article, we examine how transition analysis techniques can be utilized to monitor column integrity. A case study on the application of this method during a large scale Protein A capture step in an antibody purification process shows how it can assist with improving process knowledge and increasing the efficiency of manufacturing operations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:383–390, 2014     

Size exclusion chromatography on soft and semi-rigid packing materials in the dynamic axial compression mode

The effect of dynamic axial compression within a range of up to 5 bar upon the structure of the bed packed with soft and semi-rigid packing materials (Sephadex G-25, Bio-Gel P2 and Toyopearl HW-40) and the associated chromatographic parameters were studied for size exclusion chromatography. Continuous packing compression is accomplished by use of a special column with controlled external pressure applied to the packing. Compression has been shown to favor an overall increase in the resolution with pressure optima observed in some cases.     

Toward a robust model of packing and scale-up for chromatographic beds. 2. Flow packing

We developed and evaluated a model for predicting the flow packing of nonrigid chromatographic resins. The model is based on elasticity theory and accounts for resin rigidity and column diameter. When a modulus determined from a standard mechanical compression (consolidation) test is used, the model captures the primary phenomena of the scale-up process. However, moduli determined from flow-packing experiments improve the accuracy of the predictions and show that the apparent rigidity of chromatographic resins is lower for flow packing than for mechanical compression. Using a modulus from flow-packing experiments provided quantitative scale-up predictions of flow packing carried out in columns with diameters between 200 and 450 mm at different locations and by different operators.     

Transport and binding characterization of a novel hybrid particle impregnated membrane material for bioseparations

The transport and binding properties of a novel hybrid particle-nonwoven membrane medium are described. In this construct, a polymeric chromatographic resin is entrapped between two layers of a nonwoven polypropylene membrane. The membrane-supported resin medium offers the advantage of increased interstitial pore diameter to allow passage of cells and other debris in the feed, while providing sufficiently high surface area for product capture within the resin particles. Columns packed with PIM displayed excellent flow distribution and had interstitial porosities of 0.48 ± 0.01, 25-60% larger than those typical of a packed bed. These columns were able to pass over 95% of E. coli cells and human red blood cell concentrate in 30 column volumes while maintaining a pressure drop significantly lower than that of a packed bed with a similar amount of resin. The dynamic binding capacity of bovine serum albumin (BSA) to the chromatographic resin entrapped in the PIM packed column was essentially the same as that observed with the same volume of resin in a packed bed. The General Rate (GR) model of chromatography was used to analyze experiments indicating the breakthrough behavior of the PIM columns is predictable, and very similar to those of a normal packed bed. These results suggest that PIM constructs can be designed to process viscous mobile phases containing particulates while retaining the desirable binding characteristics of the embedded chromatographic resin and could find uses in adsorption separation processes from complex feed streams such as whole blood, cell culture, and food processing.     

Chromatography of hen egg-white proteins on anion-exchange cellulose at high flow rates using a radial flow column.

The influence of column configuration on the separation of hen egg-white proteins using Whatman DE52 and QA52 anion-exchange cellulose has been investigated. Using a 100 ml volume axial flow column (6.6 cm x 4.4 cm i.d.) we achieved flow rates of up to 25 ml/min i.e. 15 bed volumes/h after which higher flow was restricted due to pressure constraints within the system. Under radial flow conditions using a 100 ml column flow rates of up to 150 ml/min i.e. 90 bed volumes/h were achieved using DE52 and QA52. While chromatographic resolution was superior under axial flow at the lower flow rates excellent resolution was maintained at up to 150 ml/min using the radial flow column. This is a consequence of the fast kinetics of adsorption/desorption exhibited by DE52 and QA52. The data indicate that it is the column configuration and not the cellulose matrix which influences flow performance.     

Optimization Considerations for the Purification of α1-Antitrypsin Using Silica-Based Ion-Exchange Adsorbents in Packed and Expanded Beds

The influences of the fluid superficial velocity, sample concentration, loading volume, and wash cycle on the recovery and corresponding purification factors for α₁-antitrypsin [syn. α₁-proteinase inhibitor (α₁-PI) ] from crude mixtures of human plasma proteins were investigated using packed and expanded beds of DEAE-Spherodex LS. As part of this study, the effect of fluid superficial velocity on the bed dispersion number (D _v) and dispersion coefficient (D) for this adsorbent in expanded beds was determined with feedstocks containing human serum albumin (HSA), the most abundant of the contaminating proteins in human plasma protein preparations used for the isolation of α₁-PI. When multicomponent protein feedstocks prepared from human plasma were examined with DEAE-Spherodex LS, reduced chromatographic productivity was observed for α₁-PI as the extent of column utilization and the superficial velocity were increased, yet the opposite trend was evident for HSA. In particular, higher adsorption capacities and recoveries were obtained for α₁-PI at lower fluid superficial velocities with both packed and expanded bed conditions. These findings indicate that for process scale purifications of α₁-PI from multicomponent feedstocks with expanded beds containing this silica-based ion-exchange adsorbent, the optimal range of superficial velocities to achieve the highest bed productivity will not be synonymous with maximally fluidized modes of operation. Rather, the results confirm that the adsorbent has an optimum operational performance when fluidization procedures corresponding to plug flow expansion are employed for the capture of α₁-PI. These findings also indicate that advantage can be taken of displacement effects between closely related protein species with packed and expanded bed systems containing the DEAE-Spherodex LS type of ion-exchange porous silicas.     

Packing of large‐scale chromatography columns with irregularly shaped glass based resins using a stop‐flow method

Rigid chromatography resins, such as controlled pore glass based adsorbents, offer the advantage of high permeability and a linear pressure‐flow relationship irrespective of column diameter which improves process time and maximizes productivity. However, the rigidity and irregularly shaped nature of these resins often present challenges in achieving consistent and uniform packed beds as formation of bridges between resin particles can hinder bed consolidation. The standard flow‐pack method when applied to irregularly shaped particles does not yield well‐consolidated packed beds, resulting in formation of a head space and increased band broadening during operation. Vibration packing methods requiring the use of pneumatically driven vibrators are recommended to achieve full packed bed consolidation but limitations in manufacturing facilities and equipment may prevent the implementation of such devices. The stop‐flow packing method was developed as an improvement over the flow‐pack method to overcome these limitations and to improve bed consolidation without the use of vibrating devices. Transition analysis of large‐scale columns packed using the stop‐flow method over multiple cycles has shown a two‐ to three‐fold reduction of change in bed integrity values as compared to a flow‐packed bed demonstrating an improvement in packed bed stability in terms of the height equivalent to a theoretical plate (HETP) and peak asymmetry (A_s). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1319–1325, 2014     

Drug analysis by direct liquid introduction micro liquid chromatography mass spectrometry

The analytical capabilities of a micro high performance liquid chromatograph interfaced to an unchanged quadrupole mass spectrometer are presented. Continuous monitoring of the total micro liquid chromatographic effluent allows full scan chemical ionization mass spectra of from one to five nanograms of drugs and their metabolites to be recorded. The interface is a simple, inexpensive device which can be assembled from commercially available components. An eight microliter per minute flow rate of the micro liquid chromatographic eluant allows separation and identification of biologically important substances not amenable to gas chromatography mass spectrometry techniques. The sensitivity of micro liquid chromatography mass spectrometry performed as described is comparable with gas chromatography mass spectrometry and is achieved by introducing the total micro liquid chromatographic effluent into the chemical ionization ion source of the mass spectrometer. Selected ion monitoring provides 20 pg detection limits of phenothiazine tranquilizers injected on column.     

内循环颗粒污泥床硝化反应器临界曝气强度的研究

内循环颗粒污泥床硝化反应器是一种新型高效硝化反应器 ,在反应器运行过程中 ,液体循环临界曝气强度和颗粒污泥流化临界曝气强度是两个重要操作参数。建立了升流区表观液速Ulr与曝气强度Ugr之间的关系 ,并测定了有关的模型参数 ,得到了具体的数学表达式 :U_lr=(2.613-0.024 )U^0.871_gr 0.276U^0.871_gr-0.28。根据该模型 ,计算得到的液体循环临界曝气强度为1.017cm/min ,颗粒污泥流化临界曝气强度为 2.662cm/min。实测结果证明 ,求得的两个临界曝气强度具有较高的准确性 ,能够用于指导内循环颗粒污泥床硝化反应器的操作优化.     

Operational parameters for packed beds in solid-state cultivation

Packed bed cultivation systems have potential for widespread application in solid-state cultivation (SSC), but they are poorly characterized. The effects of particle size and substrate loading on the growth of Rhizopus oligosporus on sago-beads in packed bed bioreactors were investigated. Pressure drop and protein were monitored as indicators of fungal growth in cultivations performed in a large column (4.9 cm internal diameter and 60 cm height) and a system of small columns (4.2 cm internal diameter and 5.2 cm height). The differential pressure drop increased to a maximum between 34 and 44 h and then decreased again. The maximum differential pressure drop attained was greatest for the smallest particle size and for the lower substrate loadings. However, since the protein content continued to increase throughout the cultivation, pressure drop could not be used to monitor growth directly.     

Adsorption and desorption kinetics of bovine serum albumin in ion exchange and hydrophobic interaction chromatography on silica matrices

Large scale chromatographic separation of proteins can be carried out more rapidly on rigid adsorbents than on soft gel media. The kinetics of adsorption of bovine serum albumin (BSA) have been studied on rigid adsorbents based on a wide-pore, hydrophilically-coated silica gel matrix in a packed bed (chromatographic column). Process parameters have been varied comprehensively. The effects of surface chemistry (weak anion exchanger and hydrophobic interaction), particle size and liquid flow velocity have been studied on both the adsorption and desorption processes. The relative influences of the adsorption kinetics and equilibrium isotherm on the shape of the breakthrough curve are found to vary with the process parameters in an interpretable and therefore, predictable manner. Pore diffusion resistance is dominant over the external liquid film resistance in controlling the adsorption kinetics, with Biot numbers in the range 170-2600. A two-step model based on these two resistances simulates the breakthrough curves with only limited quantitative accuracy, but gives good predictions of the effect of changes in process parameters.     

Investigating the use of column inserts to achieve better chromatographic bed support

Chromatography plays an important role in the downstream processing of proteins. Over the past years, there has been a steady move toward the adoption of more rigid, porous particles to combine ease of manufacture with increased levels of productivity. The latter is still constrained by the onset of compression where the level of wall support becomes incapable of withstanding flow‐induced particle drag. In this study, we investigate how, by the installation of cylindrical column inserts, it is possible to enhance the level of wall support. Experiments were conducted to examine the effect of the position of the insert in the column, and also of the insert dimensions on the critical velocity at which the onset of compression occurs. It was found that when installed at the bottom of the column, inserts can provide up to a 20% increase in critical velocity without significantly affecting column hydrodynamics, as measured by the level of axial dispersion. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012